Uses of diterpenoid triepoxides as an antiproliferative agent

ABSTRACT

Combinations of diterpenoid triepoxides and anti-proliferative agents are used in a combination therapy to treat hyperproliferative disorders. Anti-proliferative agents of interest include agents active in killing tumor cells, as well as immunosuppressants, and a variety of other agents that reduce cellular proliferation in targeted tissues. Synergistic combinations provide for comparable or improved therapeutic effects, while lowering adverse side effects.

CROSS REFERENCE OF PRIOR APPLICATIONS

[0001] This application is a continuation-in-part of prior applicationSer. No. 09/385,917, filed Aug. 30, 1999.

BACKGROUND

[0002] Progress in the treatment of solid tumors has been slow andsporadic despite the development of new chemotherapeutic agents. Thereare many roadblocks to successful chemotherapy, including drugresistance, resistance to apoptosis, and the inactivation of tumorsuppressor genes. Some human cancers are drug resistant before treatmentbegins, while in others drug resistance develops over successive roundsof chemotherapy.

[0003] One type of drug resistance, called multidrug resistance, ischaracterized by cross resistance to functionally and structurallyunrelated drugs. Typical drugs that are affected by the multidrugresistance are doxorubicin, vincristine, vinblastine, colchicine,actinomycin D, and others. At least some multidrug resistance is acomplex phenotype that is linked to a high expression of a cell membranedrug efflux transporter called Mdr1 protein, also known asP-glycoprotein. This membrane “pump” has broad specificity and acts toremove from the cell a wide variety of chemically unrelated toxins.

[0004] Another factor in cancer therapy is the susceptibility oftargeted cells to apoptosis. Many cytotoxic drugs that kill cells bycrippling cellular metabolism at high concentration can triggerapoptosis in susceptible cells at much lower concentration. Increasedsusceptibility to apoptosis can be acquired by tumor cells as abyproduct of the genetic changes responsible for malignanttransformation, but most tumors tend to acquire other genetic lesionswhich abrogate this increased sensitivity. Either at presentation orafter therapeutic attempts, the tumor cells can become less sensitive toapoptosis than vital normal dividing cells. Such tumors are generallynot curable by conventional chemotherapeutic approaches. Althoughdecreased drug uptake, altered intracellular drug localization,accelerated detoxification and alteration of drug target are importantfactors, pleiotropic resistance due to defective apoptotic response isalso a significant category of drug resistance in cancer.

[0005] An important tumor suppressor gene is the gene encoding thecellular protein, p53, which is a 53 kD nuclear phosphoprotein thatcontrols cell proliferation. Mutations to the p53 gene and allele losson chromosome 17p, where this gene is located, are among the mostfrequent alterations identified in human malignancies. The p53 proteinis highly conserved through evolution and is expressed in most normaltissues. Wild-type p53 has been shown to be involved in control of thecell cycle, transcriptional regulation, DNA replication, and inductionof apoptosis.

[0006] Various mutant p53 alleles are known in which a single basesubstitution results in the synthesis of proteins that have quitedifferent growth regulatory properties and, ultimately, lead tomalignancies. In fact, the p53 gene has been found to be the mostfrequently mutated gene in common human cancers, and is inactivated in˜50% of tumors. It is particularly associated with those cancers linkedto cigarette smoke. The overexpression of p53 in breast tumors has alsobeen documented.

[0007] The increase in p53 protein in response to various stresses is animportant regulator of cell cycle and apoptosis. Intranscription-dependent p53 activation, p53 functions as a site-specifictranscription factor that induces p53-inducible genes such asp21cip1/waf1, bax, gadd45, and mdm2. This in turn initiates the programof growth arrest or apoptosis in a stress-specific and/or celltype-specific manner. Two recent studies have shown that p53-mediatedinduction of p21 inhibits the apoptotic response by inducing growtharrest. For example, p53- or p21-deficient cells have been shown to bemore sensitive to adriamycin (doxorubicin)-induced apoptosis thanwild-type cells because they do not induce p21 in response todoxorubicin. Therefore, a compound that blocks chemotherapy-mediatedgrowth arrest may accelerate and enhance apoptosis.

[0008] An area to search for new therapeutic interventions is that oftraditional Chinese medicines. One of these traditional medicines isfrom Tripteryguim wilfordii Hook F, a shrub-like vine from theCelastraceae family. A variety of preparations derived from this planthave been used in South China for many years to treat different forms ofarthritis and other autoimmune diseases. In 1978, an extract ofTripterygium wilfordii Hook F was produced by chloroform methanolextraction of the woody portion of the roots and designated T2. Reportsin the Chinese literature describe T2 treatment of more than 750patients with a variety of autoimmune diseases.

[0009] The Chinese experience has suggested that a daily dosage of about1 mg/kg of T2 is safe and effective as an immunosuppressant. Acute andchronic toxicity studies have been carried out in China using a varietyof animal models. The LD₅₀ in mice was reported to be around 150 mg/kg.The toxicity studies suggest that T2 exhibits a reasonable safety indexand should be able to be administered to patients safely.

[0010] The development of chemotherapeutic agents and combinations ofagents that avoid problems of drug resistance and resistance toapoptosis are of great interest for the treatment of cancer.

[0011] Literature

[0012] The isolation, purification, and characterization ofimmunosuppressive compounds from tripterygium: triptolide andtripdiolide is reported by Gu et al. (1995) Int J Immunopharmacol17(5):351-6. Yang et al. (1998) Immunopharmacology 40(2):139-49 provideevidence that suggests the immunosuppressive agent triptolide inhibitsantigen or mitogen-induced T cell proliferation, and induces apoptoticdeath of T cell hybridomas and peripheral T cells. Shamon et al. (1997)Cancer Lett 112(1):113-7 evaluate the antitumor potential of triptolide.Tengchaisri et al. (1998). Cancer Lett. 133(2):169-75 evaluate theantitumor activity of triptolide against cholangiocarcinoma growth invitro and in hamsters.

[0013] Lee et al. (1999) J Biol Chem 274(19):13451-5 describe theinteraction of PG490 (triptolide) with tumor necrosis factor-alpha toinduce apoptosis in tumor cells. Triptolide was found to inhibit T-cellinterleukin-2 expression at the level of purine-box/nuclear factor ofactivated T-cells and NF-kappa B transcriptional activation by Qiu etal. (1999) J. Biol. Chem. 274(19): 13443-50.

[0014] Chang et al. (2000) J. Biol. Chem. 276:2221-2227 describes thecooperation of triptolide with chemotherapeutic agents in tumor cellapoptosis.

SUMMARY OF THE INVENTION

[0015] Compositions and methods are provided for the use of diterpenoidtriepoxides in combination with anti-proliferative agents, as acombination therapy to treat hyperproliferative disorders. The methodsand compositions are particularly useful in the treatment of multi-drugresistant tumor cells. Anti-proliferative agents of interest includeagents active in killing tumor cells, as well as immunosuppressants, anda variety of other agents that reduce cellular proliferation in targetedtissues. The targeted cells are contacted with an anti-proliferativeagent and diterpenoid triepoxides, e.g. triptolide, tripdiolide, etc.,or prodrugs that convert to such compounds under physiologicalconditions, either locally or systemically. Synergistic combinationsprovide for comparable or improved therapeutic effects, while loweringadverse side effects.

[0016] The combination therapy is particularly effective in thetreatment of tumor cells that express p21^(waf1/cip1), which cells canotherwise be resistant to the induction of apoptosis. In one embodimentof the invention, tumors are assayed for the expression of p21 todetermine whether they will benefit from the combined therapy of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a graph depicting the cytotoxicity of PG490 in tumorcells.

[0018]FIG. 2 is a graph depicting the inhibition of PG490-inducedapoptosis.

[0019]FIG. 3 illustrates the effect of PG490-88 on preestablished H23tumors.

[0020]FIG. 4 depicts the effect of PG490-88 on preestablished Dx5 MDRtumors.

[0021]FIG. 5. Triptolide inhibits Mdm2 gene expression.

[0022]FIG. 6. PG490-88 and CPT-11 provide for a synergistic combinationin treating tumors in vivo.

[0023]FIG. 7. Combination treatment of nude mice bearing establishedHT-29 human colon cancer cell line tumors with PG490-88 and Navelbine.

[0024]FIG. 8. Combination treatment of nude mice bearing establishedHT-29 human colon cancer cell line tumors with PG490-88 and Navelbine.

[0025]FIGS. 9A and 9B. Tritolide induces p53 but inhibits p21expression. FIG. 9A, subconfluent HT180 tumor cell lines were treated asshown. After 9 h. total cellular lysate was harvested followed byimmunoblot analysis with a p53 mAb. The blot was then stripped andreprobed with a p21 mAb. FIG. 9B, MEFs (p53+/+ and p52−/−) wereimmunoblotted with a p21 mAb. The gel was scanned, and the p53 bandswere with a p21 mAb. The gel was scanned, and the p53 bands weremeasured using a densitometry program. Protein phosphatase-1 (PP1) isused as a loading control.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] Diterpenoid triepoxides are formulated in combination withanti-proliferative agents, as a combination therapy to treathyperproliferative disorders. Although the diterpenoid triepoxides, andthe anti-proliferative agents, are active when administered alone, theconcentrations required for a killing dose may create unacceptable sideeffects. The methods and compositions are particularly useful in thetreatment of multi-drug resistant tumor cells.

[0027] Anti-proliferative agents of interest include agents active inkilling tumor cells, as well as immunosuppressants, and a variety ofother agents that reduce cellular proliferation in targeted tissues. Thetargeted cells are contacted with an anti-proliferative agent andditerpenoid triepoxides, e.g. triptolide, tripdiolide, etc., or prodrugsthat convert to such compounds under physiological conditions, eitherlocally or systemically. Synergistic combinations provide for comparableor improved therapeutic effects, while lowering adverse side effects.The subject methods provide a means for therapeutic treatment andinvestigation of hyperproliferative disorders, through the induction ofa novel cell-killing pathway. Animal models, particularly small mammals,e.g. murine, lagomorpha, etc. are of interest for experimentalinvestigations.

[0028] The subject methods are used for prophylactic or therapeuticpurposes. The term “treatment” as used herein refers to reducing oralleviating symptoms in a subject, preventing symptoms from worsening orprogressing, inhibition or elimination of the causative agent, orprevention of the disorder in a subject who is free therefrom. Forexample, treatment of a cancer patient may be reduction of tumor size,elimination of malignant cells, prevention of metastasis, or theprevention of relapse in a patient whose tumor has regressed. Thetreatment of ongoing disease, to stabilize or improve the clinicalsymptoms of the patient, is of particular interest. Such treatment isdesirably performed prior to complete loss of function in the affectedtissues.

[0029] In one aspect of the invention, the targeted cell population is atumor cell population. Particular benefits are obtained when thetargeted cells express functional p53 protein. Triptolide uses the p53pathway to kill tumor cells that express wild-type p53, although itsability to kill cancer cells does not depend on p53.

[0030] Cells that express p21^(waf1/cip1) protein have a particularbenefit from the combination therapy with triptolide-based compounds andcommon chemotherapeutic agents. Activation of p53 in tumor cells, forexample, can induce expression of p21^(waf1/cip1). Expression ofp21^(waf1/cip1) can then induce growth arrest, which makes the cellsresistant to induction of apoptosis. Triptolide acts in combinationtherapy to block chemotherapy-mediated induction of p21^(waf1/cip1),resulting in an increased susceptibility to the chemotherapeutic agents.Tumor cells, e.g. biopsy samples, can be screened for expression ofp21^(waf1/cip1) either constitutively, or in response to the candidatechemotherapeutic agent, induction of p53, and the like. Patients withtumors that constitutively or inductively express p21^(waf1/cip1) may beselected for the combination therapies of the present invention.

[0031] Chemotherapeutic agents that particularly benefit from thecombined therapy are those that are susceptible to p21^(waf1/cip1)mediated resistance, and include agents that induce expression of p53,and consequently p21^(waf1/cip1) For example, without limitation,combination therapies can be formulated with the topoisomeraseinhibitors anthracyclines, including the compounds daunorubicin,adriamycin (doxorubicin) epirubicin, idarubicin, anamycin, MEN 10755,and the like. Other topoisomerase inhibitors include the podophyllotoxinanalogues etoposide and teniposide, and the anthracenediones,mitoxantrone and amsacrine. While topoisomerases are required forproliferation, proliferation is also essential for efficacioustopoisomerase inhibition, making these compounds particularly sensitiveto endogenous agents that induce growth arrest.

[0032] In one aspect of the invention, the anti-proliferative agentsinterferes with microtubule assembly, e.g. the family of vincaalkaloids. Examples of vinca alkaloids include vinblastine, vincristine;vinorelbine (NAVELBINE); vindesine; vindoline; vincamine; etc. Drugresistance to these compounds is due primarily to decreased drugaccumulation and results from overexpression of the P-glycoprotein. Themethods of the present invention may find use in preventing theselection of drug resistant cells, and in treating resistant tumors.

[0033] In one embodiment of the invention, the anti-proliferative agentis a DNA-damaging agent, such as nucleotide analogs, e.g. purines andpyrimidines, alkylating agents, etc. Another anti-proliferative agent ofparticular interest is taxol.

[0034] In another embodiment of the invention, the anti-proliferativeagent is a topoisomerase inhibitor, e.g. a topoisomerase I inhibitor ora topoisomerase II inhibitor.

Definitions

[0035] It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

[0036] Hyperproliferative disorders: refers to excess cellproliferation, relative to that occurring with the same type of cell inthe general population and/or the same type of cell obtained from apatient at an earlier time. The term denotes malignant as well asnon-malignant cell populations. Such disorders have an excess cellproliferation of one or more subsets of cells, which often appear todiffer from the surrounding tissue both morphologically andgenotypically. The excess cell proliferation can be determined byreference to the general population and/or by reference to a particularpatient, e.g. at an earlier point in the patient's life.Hyperproliferative cell disorders can occur in different types ofanimals and in humans, and produce different physical manifestationsdepending upon the affected cells.

[0037] Hyperproliferative cell disorders include cancers; blood vesselproliferative disorders such as restenosis, atherosclerosis, in-stentstenosis, vascular graft restenosis, etc.; fibrotic disorders;psoriasis; inflammatory disorders, e.g. arthritis, etc.; glomerularnephritis; endometriosis; macular degenerative disorders; benign growthdisorders such as prostate enlargement and lipomas; and autoimmunedisorders. Cancers are of particular interest, including leukemias,lymphomas (Hodgkins and non-Hodgkins), and other myeloproliferativedisorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue,hypoxic tumors, squamous cell carcinomas of the mouth, throat, larynx,and lung, genitourinary cancers such as cervical and bladder cancer,hematopoietic cancers, head and neck cancers, and nervous systemcancers, benign lesions such as papillomas, and the like.

[0038] Multidrug resistant cells: Cells of particular interest for thesubject anti-proliferative therapy are multi-drug resistant. Multi-drugresistance is frequently caused by an integral glycoprotein in theplasma membrane of the targeted cell, P-glycoprotein(pleiotropic-glycoprotein, Pgp, MDR1), or a related homolog (MRP). Whenexpressed by tumor cells, MDR1 expels cytotoxic chemotherapeutic agents,and thus allows the tumor cell to survive anticancer treatments even athigh drug doses. For example, treatment with vinca alkaloids can becompromised by the development of multidrug resistance.

[0039] Various methods may be used to determine whether a particulartumor cell sample is multi-drug resistant. Multi-drug resistance can bediagnosed in tumors by molecular biology techniques (gene expression atthe mRNA level), by immunological techniques (quantification ofP-glycoprotein itself) or by functional approaches (measuring dyeexclusion). The sequence of P-glycoprotein may be obtained as Genbankaccession number NM_(—)000927 (Chen et al. (1986) Cell 47:381-389.

[0040] In MDR1-expressing cells a decreased uptake of cytotoxic drugscan be visualized by measuring the cellular accumulation or uptake offluorescent compounds, e.g., anthracyclines (Herweijer et al. (1989)Cytometry 10:463-468), verapamil-derivatives (Lelong et al. (1991) Mol.Pharmacol. 40:490-494), rhodamine 123 (Neyfakh (1988) Exp. Cell Res.174:168-174); and Fluo-3 (Wall et al. (1993) Eur. J. Cancer29:1024-1027). Alternatively, the sample of cells may be exposed to acalcein compound; measuring the amount of calcein compound accumulatingin the specimen cells relative to control cells. Reduced calceinaccumulation in specimen cells relative to control cells indicates thepresence of multi-drug resistance in the biological specimen.

[0041] diterpenoid triepoxide sensitizing agent: compounds of interestfor use in the combination therapy include compounds having thestructure:

[0042] wherein X₁ is OH, ═O; or OR¹;

[0043] X₂ and X₃ are independently OH, OR¹ or H;

[0044] R¹ is —C(O)—Y-Z, wherein Y is a branched or unbranched C₁ to C₆alkyl or alkenyl group; and Z is COOR², NR³R^(3′), or +NR⁴R^(4′)R^(4″),where R² is a cation; R³ and R^(3′) are independently H or branched orunbranched C₁ to C₆ alkyl, hydroxyalkyl, or alkoxyalkyl, or R³ andR^(3′) taken together form a 5- to 7-member heterocyclic ring whose ringatoms are selected from the group consisting of carbon, nitrogen, oxygenand sulfur, wherein the ring atoms include 2 to 6 carbon atoms, or morenitrogen atoms, and optionally one or more oxygen or sulfur atoms, andwherein the ring is unsubstituted or is substituted with one or moregroups selected from R⁵, OR⁵, NR⁵R⁶, SR⁵, NO₂, CN, C(O)R⁵, C(O)NR⁵R⁶,and halogen (fluoro, chloro, bromo, or iodo), where R⁵ and R⁶ areindependently hydrogen, lower alkyl or lower alkenyl; and R⁴, R^(4′) andR^(4″) are independently branched or unbranched C₁ to C₆ alkyl,hydroxyalkyl or alkoxyalkyl. Examples of such molecules may be found inInternational Patent application WO98/52951, and WO97/31921, hereinincorporated by reference.

[0045] Compounds of particular interest include triptolide, tripdiolide,triptonide, tripterinin, 16-hydroxytriptolide, triptriolide, andtripchloride; as well as derivatives of triptolide, 16-hydroxytriptolideand tripdiolide (2-hydroxytriptolide) that are derivatized at one ormore hydroxyl groups. Such derivatives may be ester derivatives, wherethe attached ester substituents include one or more amino or carboxylategroups. Prodrugs of particular interest include triptolide succinatesodium salt and triptolide succinate tris(hydroxymethyl)aminomethanesalt.

[0046] The compounds of the invention may be prepared from triptolide,tripdiolide, or 16-hydroxytriptolide obtained from the root xylem of theChinese medicinal plant Tripterygium wilfordii or from other knownsources. Methods for preparing triptolide and related compounds areknown in the art.

[0047] Anti-proliferative agents: agents that act to reduce cellularproliferation are known in the art and widely used. Such agents includealkylating agents, such as nitrogen mustards, e.g. mechlorethamine,cyclophosphamide, melphalan (L-sarcolysin), etc.; and nitrosoureas, e.gcarmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU),streptozocin, chlorozotocin, etc. Such agents are used in the treatmentof cancer, as well as being immunosuppressants and anti-inflammatoryagents.

[0048] Antimetabolite agents include pyrimidines, e.g. cytarabine(CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine(FUdR), etc.; purines, e.g. thioguanine (6-thioguanine), mercaptopurine(6-MP), pentostatin, fluorouracil (5-FU) etc.; and folic acid analogs,e.g. methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, etc. Methotrexateis widely used as an immunosuppressant, particularly with allogeneicorgan transplants, as well as in the treatment of otherhyperproliferative disorders. Leucovorin is useful as an anti-infectivedrug.

[0049] Other natural products include azathioprine; brequinar; alkaloidsand synthetic or semi-synthetic derivatives thereof, e.g. vincristine,vinblastine, vinorelbine, etc.; podophyllotoxins, e.g. etoposide,teniposide, etc.; antibiotics, e.g. anthracycline, daunorubicinhydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin,doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizonebiscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g.bleomycin; anthraquinone glycosides, e.g. plicamycin (mithrmycin);anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g.mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506(tacrolimus, prograf), rapamycin, etc.; and the like.

[0050] Hormone modulators include adrenocorticosteroids, e.g.prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide. Estrogens stimulate proliferationand differentiation, therefore compounds that bind to the estrogenreceptor are used to block this activity. Corticosteroids may inhibit Tcell proliferation.

[0051] Other chemotherapeutic agents include metal complexes, e.g.cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; andhydrazines, e.g. N-methylhydrazine. Other anti-proliferative agents ofinterest include immunosuppressants, e.g. mycophenolic acid,thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine,azaspirane (SKF 105685), etc., taxols, e.g. paclitaxel, etc.

[0052] Retinoids, e.g. vitamin A, 13-cis-retinoic acid, trans-retinoicacid, isotretinoin, etc.; carotenoids, e.g. beta-carotene, vitamin D,etc. Retinoids regulate epithelial cell differentiation andproliferation, and are used in both treatment and prophylaxis ofepithelial hyperproliferative disorders.

[0053] Angiotensinase inhibitors diminish exposure of the mesangium toprotein factors that stimulate mesangial cell proliferation, and areuseful with respect to vascular proliferative disorders.

[0054] An agent of particular interest for the present methods isirinotecan (CPT-11), a topoisomerase I inhibitor. CPT-11 finds use as aco-therapeutic agent, e.g. in the treatment of solid tumors, such ascolon cancer, sarcomas, non-small cell lung carcinoma, ovarian andendometrial carcinomas, adenocarcinomas, mesotheliomas, etc. Othertopoisomerase inhibitors of interest in the subject methods includedoxorubicin and carboplatinum, which inhibit type II topoisomerase.

[0055] Pharmaceutical Formulations: The diterpenoid triepoxides, and theanti-proliferative agents can be incorporated into a variety offormulations for therapeutic administration. The diterpenoid triepoxideand anti-proliferative agent can be delivered simultaneously, or withina short period of time, by the same or by different routes. In oneembodiment of the invention, a co-formulation is used, where the twocomponents are combined in a single suspension. Alternatively, the twomay be separately formulated.

[0056] Part of the total dose may be administered by different routes.Such administration may use any route that results in systemicabsorption, by any one of several known routes, including but notlimited to inhalation, i.e. pulmonary aerosol administration;intranasal; sublingually; orally; and by injection, e.g. subcutaneously,intramuscularly, etc.

[0057] More particularly, the compounds of the present invention can beformulated into pharmaceutical compositions by combination withappropriate pharmaceutically acceptable carriers or diluents, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. As such, administration of the compounds can be achieved invarious ways, including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, etc.,administration. The active agent may be systemic after administration ormay be localized by the use of regional administration, intramuraladministration, or use of an implant that acts to retain the active doseat the site of implantation.

[0058] In pharmaceutical dosage forms, the compounds may be administeredin the form of their pharmaceutically acceptable salts. They may also beused in appropriate association with other pharmaceutically activecompounds. The following methods and excipients are merely exemplary andare in no way limiting.

[0059] For oral preparations, the compounds can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

[0060] The compounds can be formulated into preparations for injectionsby dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

[0061] The compounds can be utilized in aerosol formulation to beadministered via inhalation. The compounds of the present invention canbe formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

[0062] Furthermore, the compounds can be made into suppositories bymixing with a variety of bases such as emulsifying bases orwater-soluble bases. The compounds of the present invention can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

[0063] Unit dosage forms for oral or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore compounds of the present invention. Similarly, unit dosage formsfor injection or intravenous administration may comprise the compound ofthe present invention in a composition as a solution in sterile water,normal saline or another pharmaceutically acceptable carrier.

[0064] Implants for sustained release formulations are well-known in theart. Implants are formulated as microspheres, slabs, etc. withbiodegradable or non-biodegradable polymers. For example, polymers oflactic acid and/or glycolic acid form an erodible polymer that iswell-tolerated by the host. The implant containing the therapeutic agentis placed in proximity to the site of the tumor, so that the localconcentration of active agent is increased relative to the rest of thebody.

[0065] The term “unit dosage form”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

[0066] Pharmaceutically acceptable excipients, such as vehicles,adjuvants, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.

[0067] Dosage: The combined used of diterpenoid triepoxides andanti-proliferative agents has the advantages that the required dosagesfor the individual drugs is lower, and the effect of the different drugscomplementary. Depending on the patient and condition being treated andon the administration route, the diterpenoid triepoxides will generallybe administered in dosages of 0.001 mg to 5 mg/kg body weight per day.The range is broad, since in general the efficacy of a therapeuticeffect for different mammals varies widely with doses typically being20, 30 or even 40 times smaller (per unit body weight) in man than inthe rat. Similarly the mode of administration can have a large effect ondosage. Thus for example oral dosages in the rat may be ten times theinjection dose. The dosage for the anti-proliferative agent will varysubstantially with the compound, in accordance with the nature of theagent. Higher doses may be used for localized routes of delivery.

[0068] A typical dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

[0069] Those of skill will readily appreciate that dose levels can varyas a function of the specific compound, the severity of the symptoms andthe susceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

[0070] Susceptible tumors: The host, or patient, may be from anymammalian species, e.g. primate sp., particularly humans; rodents,including mice, rats and hamsters; rabbits; equines, bovines, canines,felines; etc. Animal models are of interest for experimentalinvestigations, providing a model for treatment of human disease.

[0071] Tumors of interest include carcinomas, e.g. colon, prostate,breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa,invasive oral cancer, non-small cell lung carcinoma, transitional andsquamous cell urinary carcinoma, etc.; neurological malignancies, e.g.neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhoodacute leukemia, non-Hodgkin's lymphomas, and other myeloproliferativedisorders, chronic Iymphocytic leukemia, malignant cutaneous T-cells,mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoidpapulosis, T-cell rich cutaneous lymphoid hyperplasia, bullouspemphigoid, discoid lupus erythematosus, lichen planus, etc.; and thelike.

[0072] Some cancers of particular interest include non-small cell lungcarcinoma. Non-small cell lung cancer (NSCLC) is made up of threegeneral subtypes of lung cancer. Epidermoid carcinoma (also calledsquamous cell carcinoma) usually starts in one of the larger bronchialtubes and grows relatively slowly. The size of these tumors can rangefrom very small to quite large. Adenocarcinoma starts growing near theoutside surface of the lung and may vary in both size and growth rate.Some slowly growing adenocarcinomas are described as alveolar cellcancer. Large cell carcinoma starts near the surface of the lung, growsrapidly, and the growth is usually fairly large when diagnosed. Otherless common forms of lung cancer are carcinoid, cylindroma,mucoepidermoid, and malignant mesothelioma.

[0073] The majority of breast cancers are adenocarcinomas subtypes.Ductal carcinoma in situ is the most common type of noninvasive breastcancer. In DCIS, the malignant cells have not metastasized through thewalls of the ducts into the fatty tissue of the breast. Infiltrating (orinvasive) ductal carcinoma (IDC) has metastasized through the wall ofthe duct and invaded the fatty tissue of the breast. Infiltrating (orinvasive) lobular carcinoma (ILC) is similar to IDC, in that it has thepotential metastasize elsewhere in the body. About 10% to 15% ofinvasive breast cancers are invasive lobular carcinomas.

[0074] Melanoma is a malignant tumor of melanocytes. Although mostmelanomas arise in the skin, they also may arise from mucosal surfacesor at other sites to which neural crest cells migrate. Melanoma occurspredominantly in adults, and more than half of the cases arise inapparently normal areas of the skin. Prognosis is affected by clinicaland histological factors and by anatomic location of the lesion.Thickness and/or level of invasion of the melanoma, mitotic index, tumorinfiltrating lymphocytes, and ulceration or bleeding at the primary siteaffect the prognosis. Clinical staging is based on whether the tumor hasspread to regional lymph nodes or distant sites. For disease clinicallyconfined to the primary site, the greater the thickness and depth oflocal invasion of the melanoma, the higher the chance of lymph nodemetastases and the worse the prognosis. Melanoma can spread by localextension (through lymphatics) and/or by hematogenous routes to distantsites. Any organ may be involved by metastases, but lungs and liver arecommon sites.

Methods of Use

[0075] A combined therapy of diterpenoid triepoxide compounds andanti-proliferative agents is administered to a host suffering from ahyperproliferative disorder. Administration may be topical, localized orsystemic, depending on the specific disease. The compounds areadministered at a combined effective dosage that over a suitable periodof time substantially reduces the cellular proliferation, whileminimizing any side-effects. Where the targeted cells are tumor cells,the dosage will usually kill at least about 25% of the tumor cellspresent, more usually at least about 50% killing, and may be about 90%or greater of the tumor cells present. It is contemplated that thecomposition will be obtained and used under the guidance of a physicianfor in vivo use.

[0076] To provide the synergistic effect of a combined therapy, thediterpenoid triepoxide active agents can be delivered together orseparately, and simultaneously or at different times within the day. Inone embodiment of the invention, the diterpenoid triepoxide compoundsare delivered prior to administration of the anti-proliferative agents.

[0077] The susceptibility of a particular tumor cell to killing with thecombined therapy may be determined by in vitro testing, as detailed inthe experimental section. Typically a culture of the tumor cell iscombined with a combination of a anti-proliferative agents and aditerpenoid triepoxide at varying concentrations for a period of timesufficient to allow the active agents to induce cell killing. For invitro testing, cultured cells from a biopsy sample of the tumor may beused. The viable cells left after treatment are then counted.

[0078] The dose will vary depending on the specific anti-proliferativeagents utilized, type of cells targeted by the treatment, patientstatus, etc., at a dose sufficient to substantially ablate the targetedcell population, while maintaining patient viability. In some casestherapy may be combined with stem cell replacement therapy toreconstitute the patient hematopoietic function.

Assays for Expression of p21

[0079] As previously described, cells that constitutively or inductivelyexpress p21 may particularly benefit from the methods and compositionsof the present invention. The expression of p21 can therefore be used ina screening assay to determine whether a particular tumor will benefitfrom a combined therapy. Sequences of the p21 protein are known in theart and publicly available. The p21 protein is also referred to ascyclin dependent kinase inhibitor 1A, waf1, or cip1. The sequence of thehuman protein and corresponding genetic sequence may be found inGenbank, accession number L25610, as described by Harper et al. (1993)Cell 75:805-816.

[0080] Formats for patient sampling include time courses that follow theprogression of disease, comparisons of different patients at similardisease stages, e.g. early onset, acute stages, recover stages, etc.;tracking a patient during the course of response to therapy, includingdrug therapy, vaccination and the like. Data from animals, e.g. mouse,rat, rabbit, monkey, etc. may be compiled and analyzed in order toprovide databases detailing the course of disease, etc.

[0081] Biological samples from which cells may be collected will usuallycomprise tumor cells, and may include biopsy samples, e.g. tissue fromsites of solid tumors; and in patients having metastatic tumors orleukemias, lymphomas, etc. may include blood and derivatives therefrom.The cells may be free of chemotherapeutic agents, or may be exposed to achemotherapeutic agent of interest, either in vivo from previoustherapy, or in vitro in conjunction with the assays of the invention.Generally assays will include various negative and positive controls, asknown in the art. These may include positive controls of “spiked”samples, patients with known disease, and the like. Negative controlsinclude samples from normal patients, tissue matched controls, and thelike.

[0082] In one screening method, the test sample is assayed at theprotein level. Diagnosis can be accomplished using any of a number ofmethods to determine the absence or presence or altered amounts of a p21in the test sample. For example, detection can utilize staining of cellsor histological sections (e.g. from a biopsy sample) with labeledantibodies, performed in accordance with conventional methods. Cells canbe permeabilized to stain cytoplasmic molecules. In general, antibodiesthat specifically bind p21 are added to a sample, and incubated for aperiod of time sufficient to allow binding to the epitope, usually atleast about 10 minutes. The antibody can be detectably labeled fordirect detection (e.g., using radioisotopes, enzymes, fluorescers,chemiluminescers, and the like), or can be used in conjunction with asecond stage antibody or reagent to detect binding (e.g., biotin withhorseradish peroxidase-conjugated avidin, a secondary antibodyconjugated to a fluorescent compound, e.g. fluorescein, rhodamine, Texasred, etc.) The absence or presence of antibody binding can be determinedby various methods, including flow cytometry of dissociated cells,microscopy, radiography, scintillation counting, etc. Any suitablealternative methods of qualitative or quantitative detection of levelsor amounts of differentially expressed polypeptide can be used, forexample ELISA, western blot, immunoprecipitation, radioimmunoassay, etc.

[0083] Any suitable qualitative or quantitative methods known in the artfor detecting specific mRNAs can also be used. mRNA can be detected, forexample, by hybridization to a microarray, in situ hybridization intissue sections, by reverse transcriptase-PCR, or: in Northern blotscontaining poly A⁺ mRNA. One of skill in the art can readily use thesemethods to determine expression of p21 mRNA. Any suitable method fordetecting and comparing mRNA expression levels in a sample can be usedin connection with the methods of the invention.

[0084] Alternatively, gene expression in a test sample can be performedusing serial analysis of gene expression (SAGE) methodology (Velculescuet al., Science (1995) 270:484). SAGE involves the isolation of shortunique sequence tags from a specific location within each transcript.The sequence tags are concatenated, cloned, and sequenced. The frequencyof particular transcripts within the starting sample is reflected by thenumber of times the associated sequence tag is encountered with thesequence population.

[0085] Alternatively, gene expression in a sample using hybridizationanalysis, which is based on the specificity of nucleotide interactions.Oligonucleotides or cDNA can be used to selectively identify or captureDNA or RNA of specific sequence composition, and the amount of RNA orcDNA hybridized to a known capture sequence determined qualitatively orquantitatively, to provide information about the relative representationof a particular message within the pool of cellular messages in asample. Hybridization analysis can be designed to allow for concurrentscreening of multiple samples for the relative expression of p21.

[0086] Methods for collection of data from hybridization of samples witharrays are also well known in the art. For example, the polynucleotidesfrom the cell samples can be generated using a detectable fluorescentlabel, and hybridization of the polynucleotides in the samples detectedby scanning the hybridizations for the presence of the detectable label.Methods and devices for detecting fluorescently marked targets ondevices are known in the art. Generally, such detection devices includea microscope and light source for directing light at a substrate. Aphoton counter detects fluorescence from the substrate, while an x-ytranslation stage varies the location of the substrate.

[0087] Methods for analyzing the data collected from hybridizations arewell known in the art. For example, where detection of hybridizationinvolves a fluorescent label, data analysis can include the steps ofdetermining fluorescent intensity as a function of substrate positionfrom the data collected, removing outliers, i.e. data deviating from apredetermined statistical distribution, and calculating the relativebinding affinity of the targets from the remaining data. The resultingdata can be displayed as an image with the intensity in each regionvarying according to the binding affinity between targets and probes.

[0088] Tumor cells that induce p21 expression in response to achemotherapeutic agent of interest, or that express p21 constitutively,are considered to be particularly suitable candidates for the combinedtherapy of the present invention.

[0089] It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

[0090] As used herein the singular forms “a”, “and”, and “the” includeplural referents unless the context clearly dictates otherwise. Thus,for example, reference to “a cell” includes a plurality of such cellsand reference to “the array” includes reference to one or more arraysand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

[0091] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, thecell lines, constructs, and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

[0092] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the subject invention, and are not intended to limitthe scope of what is regarded as the invention. Efforts have been madeto ensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXPERIMENTAL Example 1 In Vivo Antitumor Activity of a Derivative ofTriptolide

[0093] Materials and Methods

[0094] Cells and Transfections. H23 (non-small cell lung cancer) andZR-75 (breast cancer) cell lines were purchased from ATCC. The Bcl-2expression vector was provided by Fred Hutchinson Cancer ResearchCenter, Seattle, Wash. MES-SA and Dx5 cell lines were provided byBranimir Sikic (Stanford University). Cells were cultured in theappropriate medium with 10% FCS supplemented with L-glutamine,penicillin and streptomycin. To examine the effect of Bcl-2 on cellsurvival, the Bcl-2 expression vector or the vector alone wasco-transfected with a β galactosidase expression vector (Invitrogen,Carlsbad, Calif.) at a 5:1 ratio using lipofectamine plus (GIBCO BRL,Gaithersburg, Md.) into Dx5 cells. After 36 h cells were stained with5-bromo-4-chloro-3-indolyl □-D-galactopyranoside (X-gal). Cell survivalwas calculated as number of total cells-blue cells/total number of cellsin a 90 mm² area from duplicate plates and expressed as the mean±S.D.

[0095] Cell death reagents and assays. Cell viability was measured by anMTT assay as recently described (Lee et al. (1999) J. Biol. Chem.274:13451-13455. z-VAD-fluoromethylketone (z-VAD.fmk) was obtained fromAlexis Biochemicals, San Diego, Calif. The effect of z-VAD.fmk on cellviability was analyzed by annexin and propidium iodide staining followedby FACS analysis according to the manufacturer's protocol (ClontechLaboratories, Palo Alto, Calif.). The analysis of apoptosis inhistologic sections was done by terminal deoxynucleotidyl transferase(TdT)-mediated d-UTP nick end labeling (TUNEL) of slides from paraffinsections of day 3 tumors harvested from the mice 24 h after the secondof two daily treatments with PG490-88 or saline. TUNEL staining was doneaccording to the manufacturer's protocol (Boehringer Mannheim,Indianapolis, Ind.) and then the histology slides were counterstainedwith methyl green. DNA was isolated from cells for analysis ofinternucleosomal DNA laddering followed by agarose gel electrophoresisand ethidium bromide staining.

[0096] Purification of PG490 and PG490-88. PG490 (triptolide) iscomposed of white to off-white crystals, has a melting point of 226-240°C., produces a single spot on thin layer chromatography, conforms to astandard triptolide preparation by Proton Nuclear Magnetic Resonance, is97% pure by reverse phase HPLC evaluation usingacetonitrile:water:methanol, and is within 0.4% of the theoreticalresult for elemental analysis (66.51% C, 6.43% H compared to thetheoretical values of 66.65% C, 6.71% H).

[0097] PG490-88, 14-succinyl triptolide sodium salt preparedsemisynthetically from PG490, is composed of white amorphous powder, hasa melting point of 232-250° C., produces a single spot on thin layerchromatography, conforms to a standard PG490-88 preparation by ProtonNuclear Magnetic Resonance, and is 98% pure by reverse phase HPLCevaluation using acetonitrile:methanol:0.006M sodium phosphate pH=3.2.PG490-88 is a prodrug of PG490, with a half-life in mouse serum of <5min at room temperature. Stock solutions of PG490-88 (1 mg/ml) wereprepared by dissolution in 0.9% NaCl and sterilized by microfiltrationusing 0.2 μm pore size filters (Supor Acrodisc 25, Gelman Sciences, AnnArbor, Ml). The PG490-88 stock solutions were diluted in 0.9% NaCl forIP administration.

[0098] Doxorubicin (Gensia Laboratories, Ltd., Irvine, Calif.) purchasedas a stock solution of 200 mg/ml was prepared for IP administration bydilution in 0.9% NaCl. Taxol was prepared by dissolution in ethanol andaddition of an equal volume of cremophor EL (Sigma, St. Louis, Mo.) toproduce a stock solution of 30 mg/ml, which was diluted in 0.9% NaCl forIP administration.

[0099] Nude mouse xenograft model. Female NCr nude mice were purchasedfrom Taconic, Germantown, N.Y., and were generally 20-24 grams whenused. Mice were kept in autoclaved filter-top microisolator cages withautoclaved water and sterile food ad lib. The cages were maintained inan isolator unit providing filtered air (Lab Products, Inc., Maywood,N.J.). Tumor cells were grown and harvested as described above. NCr nudemice were injected intradermally with 5×10⁶ tumor cells. In someexperiments, treatment was initiated on the day of tumor cellimplantation. Otherwise, tumor size was monitored, the mice were groupedtogether to constitute a similar mean tumor size in each group in anexperiment, and treatment was initiated. Mice were treated IP daily for5 days per week.

[0100] Results

[0101] PG490 (triptolide) induces apoptosis in tumor cells in vitro.PG490 alone was found to be cytotoxic on tumor cell lines which includeH23 cells, a non-small cell lung cancer cell line with mutant p53, Dx5cells, an MDR uterine sarcoma cell line derived from the MES-SA parentcell line and ZR-75 cells, a breast cancer cell line. Dx5 cells are100-fold more resistant to doxorubicin and 1000-fold more resistant totaxol than the MES-SA parent cell line (Chen et al. (1994) Cancer Res.54:4980-4987). PG490 at a dosage of 10 ng/ml decreased cell viability by65-70% of cells in the H23 and Dx5 cell lines and by 24% of cells in theZR-75 cell line. PG490 at 20 ng/ml reduced cell viability by greaterthan 80% in all three cell lines (FIG. 1).

[0102] In FIG. 1A, ZR-75 (breast cancer), H23 (non-small cell lungcancer) and Dx5 (MDR uterine sarcoma) cell lines were treated with PG490at dosages shown and harvested 48 h later for analysis of cell viabilityby an MTT assay. Data is the mean of three experiments±S.D. In FIG. 1B,DNA was isolated from untreated or PG490-treated cells 16 h after theaddition of PG490 followed by agarose gel electrophoresis and ethidiumbromide staining.

[0103] No significant difference in sensitivity to PG490 was observedbetween the Dx5 cell line and its parent MES-SA cell line. To confirmthat PG490-induced cell death was apoptotic, the presence of PG490induced DNA laddering in Dx5 cells was examined, and it was found thatPG490 induced DNA laddering in Dx5 cells which began at 6 h and wasmaximal by 16 h.

[0104] PG490 (triptolide) did not cause growth arrest or significantlyaffect cell cycle progression in Dx5 and H23 cells. Overexpression ofBcl-2 was observed to increase the cell survival in PG490-treated Dx5cells from 15% to 72% (FIG. 2). z-VAD.fmk (100 □M), a tetrapeptidecaspase inhibitor, also increased cell viability in PG490-treated Dx5cells from 15% to 68% (FIG. 2). Bcl-2 or vector control was transientlytransfected into Dx5 cells followed by the addition of PG490 (20 ng/ml)and stained 36 h later with X-gal. % cell survival was calculated astotal cells-blue cells/total cells x 100. z-VAD.fmk (100 □M) was addedto Dx5 cells 1 h prior to the addition of PG490 (20 ng/ml) and cellswere harvested for analysis of cell viability 36 h later by annexin andpropidium iodide staining followed by FACS analysis. Data represents themean of three replicates from two independent experiments±S.D.

[0105] PG490-88 prevents human tumor development in nude mice. Theresults reported above show cytotoxicity of PG490 on tumor cells invitro. To extend these studies to an in vivo setting using human tumorcell xenografts, PG490-88 was used, a more easily administered, watersoluble prodrug of PG490. H23 tumor cells were implanted intradermallyin nude mice and the animals were left untreated or were injected IPdaily with PG490-88 starting at the time of implantation. Tumors arosein {fraction (5/5)} of the untreated mice but no tumors were observedafter 5 or 7 weeks of dosing with PG490-88 at doses ranging from 0.25 to0.75 mg/kg/day (Table 1). PG490-88 treatment was stopped after week 5 in3 mice per group and was continued for an additional 2 weeks in 2 miceper group. A visible tumor arose during the sixth week in one animal ineach group in which PG490-88 dosed at 0.5 mg/kg/day or less was stoppedbut no more visible tumors appeared in these groups after week 6 (Table1). No visible tumors developed in any of the mice through the 10 weeksof observation in mice which received 0.75 mg/kg/day of PG490-88. TABLE1 PG490-88 Treatment of Nude Mice Prevents Formation of Human TumorXenografts Number of mice in group with a tumor WEEK 5 Week 6 Week 10Untreated 5/5 5/5 5/5 PG490-88 (mg/kg/day) 0.25 0/5 1/5 1/5 0.375 0/51/5 1/5 0.5 0/5 1/5 1/5 0.75 0/5 0/5 0/5

[0106] Nude mice were implanted with H23 tumor cells (day 0). Mice wereleft untreated, or were injected IP with PG490-88 daily from the day oftumor cell implantation for 5 consecutive days per week. PG490-88 wasadministered for 5 weeks. The untreated group consisted of 5 mice. Threemice in each of the treatment groups received PG490-88 for 5 weeks, and2 mice in each of these groups were given PG490-88 for 2 additionalweeks (7 weeks total). The tumor appeared only in mice in whichtreatment had been stopped after 5 weeks.

[0107] PG490-88 inhibits the growth of established tumors of H23 humantumor cells and displays enhanced efficacy in combination therapy withtaxol. H23 tumor cells were implanted intradermally in nude mice. Whenthe tumors reached approximately 100 mm³, daily IP treatment withPG490-88 was initiated. PG490-88 inhibited tumor growth in adose-dependent manner (FIG. 3). The data in FIG. 3 represents themeasurement of H23 tumor volume on day 14 after the initiation oftreatment. Nude mice bearing xenografts of H23 human tumor cells weretreated daily as shown. The data represent the means and the standarderrors of the means of the tumor volumes as percent of the day 0 tumorvolumes for each animal measured day 3, 6, 10 and 14 days after theinitiation of treatment. There were 5 mice per group.

[0108] By day 14, the 0.25 mg/kg/day dose of PG490-88 reduced tumorvolume to 21% of the volume of the vehicle control. PG490-88 at 0.75mg/kg/day progressively reduced the mean tumor volume from day 3 throughday 14, decreasing the mean tumor size by 61% from the initial value atday 0 and a decrease of 97% relative to the day 14 vehicle control (FIG.3). Taxol decreased tumor growth at the higher dose (10 mg/kg/day) butnot the lower dose (5 mg/kg/day), with a day 14 mean tumor volume 42% ofthe vehicle control (FIG. 3). PG490-88 at 0.25 mg/kg/day plus 10mg/kg/day of taxol decreased tumor size by 93% relative to the day 14vehicle control volume (FIG. 3). Taxol at 15 mg/kg/day was not usedbecause of toxicity.

[0109] A histologic section of an H23 tumor three days after treatmentwith PG490-88 showed many cells with abundant eosinophilic cytoplasm,pyknotic nuclei with thinning or loss of nuclear membrane and condensedchromatin compared to a pattern of more homogenous spindle-shaped cellswith an increased nuclear:cytoplasmic ratio in the saline-treatedcontrol. Also, many TUNEL-positive cells were seen in thePG490-88-treated group in comparison to saline-treated animals. At day15 after the initiation of treatment with PG490-88, the H23 tumor wasreplaced by fibrous scar tissue with a central area of calcium phosphateprecipitation but the saline-treated control was unchanged in appearancecompared to the day 3 saline-treated control.

[0110] PG490-88 inhibits the growth of established tumors of an MDRhuman tumor cell line. MDR is a factor in failing to achieve durablechemotherapeutic efficacy in the clinical setting. Using an MDR tumorcell line Dx5 the efficacy of PG490-88 was tested. Nude mice wereimplanted intradermally with Dx5 tumor cells, and treatment wasinitiated when the tumors reached approximately 100 mm³ The mean tumorvolume increased more than 10-fold over the 14 days from the beginningof treatment in the groups of mice receiving saline or doxorubicin aloneat 2 mg/kg/day (FIG. 4).

[0111] The data in FIG. 4 represents the measurement of Dx5 tumor volumeon day 14 after the initiation of treatment. Nude mice bearingxenografts of Dx5 MDR human tumor cells were treated daily as shown. Thedata represent the means and the standard errors of the means of thetumor volumes as percent of the day 0 tumor volumes for each animalmeasured day 3, 7, 10 and 14 days after the initiation of treatment.There were 5 mice in the groups receiving saline or PG490-88 plusdoxorubicin, and 4 mice in the groups receiving PG490-88 or doxorubicinalone.

[0112] PG490-88 at 0.75 mg/kg/day reduced the mean tumor size by 28% inthree of the four mice compared to the day 0 values. One tumor grew by2.8-fold compared with its day 0 value. By day 14, combination treatmentwith PG490-88 and doxorubicin produced a 34% reduction in tumor volumefrom day 0 and a 94% reduction in mean tumor volume relative to the day14 vehicle control volume, with all of the tumors decreasing in sizecompared to the day 0 values.

[0113] The in vivo studies described above used PG490-88, a succinatesalt prodrug of triptolide which is rapidly converted to triptolide inthe serum. The dosage of triptolide, based on a molar comparison toPG490-88, was 70 μg/mouse/week and it was well tolerated. It wasobserved that PG490-88 at a dosage of 0.75 mg/kg completely preventedH23 tumor formation in all mice and tumors did not emerge in any of themice 5 weeks after dosing with PG490-88 was stopped.

[0114] PG490-88 also markedly inhibited the growth of preestablished H23tumors and induced apoptotic cell death in the tumor cells.Additionally, the combination of PG490-88 (0.25 mg/kg) plus taxol (10mg/kg) was more tumoricidal than either agent alone in preventing tumorformation by H23 cells. In preestablished tumors derived from the MDRDx5 cell line PG490 markedly inhibited tumor growth and doxorubicin didnot interfere with the tumoricidal activity of PG490-88. There was noobservable toxicity in mice treated with PG490-88 (0.75 mg/kg) asmeasured by a change in body weight, altered activity or laboredrespiration.

[0115] There has been progress in the treatment of some solid tumors butsignificant increases in long term survival have been limited by thedevelopment of p53 mutant and multidrug resistant tumors and by thetoxicity of chemotherapy. The above results demonstrate that PG490-88alone is a safe and potent tumoricidal agent in vivo against a p53mutant and an MDR tumor, and that the tumoricidal activity of PG490-88is enhanced by treatment with chemotherapeutic agents such as taxol.

Example 2 Triptolide Induces Apoptosis in Solid Tumor Cells and EnhancesChemotherapy-Induced Apoptosis

[0116] p53 plays a role in triptolide-induced apoptosis in tumor celllines. Also, triptolide enhances apoptosis induced by DNA-damagingchemotherapeutic agents through the p53 pathway. However, thetriptolide-mediated increase in p53 results in repression of mdm2 andp21^(Cip1/Waf1) transcription. In addition, the levels of the Mdm2 andp21 protein in triptolide-treated cells decrease late after the additionof triptolide. Interestingly, triptolide induces translation of p53without initially affecting p53 protein stability. These findingsdemonstrate that triptolide-induced apoptosis and its enhancement ofchemotherapy-induced apoptosis in p53 wild-type cells are mediated, atleast in part, by the induction of p53 translation.

[0117] Material and Methods

[0118] Reagents. PG490 (triptolide, MW 360) was obtained fromPharmagenesis (Palo Alto, Calif.). A549 (non-small cell lung cancer) andHT1080 (fibrosarcoma) cell lines were from ATCC. MCF-7 (breast cancer)cell line was obtained from Dr. Ron Weigel (Stanford University). Mouseembryonic fibroblasts (p53+/+ and p53−/−) cell lines were provided byDr. Amato J. Giaccia (Stanford University). Doxorubicin, cycloheximide,and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)were obtained from Sigma Chemicals. The mdm2 promoter-luciferaseconstruct pBP100-GL2 was provided by Dr. Louis Noumovski (StanfordUniversity) and was made by cloning the Bgl II-Hind III fragment fromthe pBP100CAT vector into the pGL2-Basic Vector (Promega, Madison,Wis.). MCF-7 cells were transfected using lipofectamine Plus reagentfrom the Life Technologies, Inc. Cells were collected and lysates wereprepared according to the manufacturer's protocol for luciferase assay(Promega Corp., Madison, Wis.). Antibodies for p53, p21^(WAF1/CIP1),Mdm2, Protein phosphatase-1 (PP-1), and Erk-2 were from Calbiochem, Inc(La Jolla, Calif.) and the rabbit polyclonal Bax antibody was fromUpstate Biotechnology (Lake Placid, N.Y.).

[0119] Cell culture and luciferase assay. A549 (non-small cell lungcancer), HT-1080 (fibrosarcoma), and MCF-7 (breast cancer) cells werecultured in the appropriate media with 10% FCS supplemented withL-glutamine, penicillin, and streptomycin. p53 wild-type (+/+) and null(−/−) Mouse Embryonic Fibroblasts (MEFs) transfected with the E1A/Raswere grown in DMEM containing 15% FCS supplemented with L-glutamine,penicillin, and streptomycin. Transfections were done on MCF-7 cellsusing the lipofectamine Plus reagent. At 24 hours after transfection,MCF-7 cells were left untreated or treated with triptolide (20 ng/ml) ordoxorubicin (100 nM) for 4, 8, and 16 hours and cells were collected forluciferase assay. Luciferase activity was measured in samples with equalprotein concentration with a Luminometer (Analytical LuminescenceLaboratory, San Diego, Calif.).

[0120] Cell viability assay. Cell viability was measured by an3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayas described above. Untreated or cells treated with triptolide and/ordoxorubicin were harvested at the indicated times followed by theaddition of MTT to the cells in a 96-well plate. Cells were solubilizedwith CH₃Cl acidified with 0.1N HCl. The 96-plate was read at awavelength of 590 nm on an iEMS Labsystems plate reader.

[0121] RT-PCR. RNA was prepared from MCF-7 cells using Rneasy Mini Kitfrom Qiagen Inc. (Valencia, Calif.). cDNAs were prepared using M-MLVreverse transcriptase (Gibco) with 2 μg of total RNA. {fraction (1/20)}of total cDNA was used in limited (25 cycles) PCR reactions using Taqpolymerase (Gibco). The following primer pairs were used: p53 [SEQ IDNO:1] 5′-AGTCAGATCCTAGCGTCGAG-3′ and 5′-[SEQ ID NO:2]TCTTCTTTGGCTGGGGAGAG-3′, mdm2, [SEQ ID NO:3]5′-GTCAATCAGCAGGAATCATCGG-3′ and [SEQ ID NO:4]5′-CAATCAGGAACATCAAAGCCCTC-3′, p21, [SEQ ID NO:5]5′-AGTGGGGCATCATCAAAAAC-3′ and [SEQ ID NO:6]5′-GACTCCTTGTTCCGCTGCTAATC-3′, and glyceraldehyde-3-phosphatedehydrogenase (GAPDH)-[SEQ ID NO:7] 5′-CCCATCACCATCTTCCAG-3′ and [SEQ IDNO:8] 5′-ATGACCTTGCCCACAGCC-3′.

[0122] Immunoblotting. At 8 hours after triptolide and/or doxorubicintreatment cells were harvested at the times indicated and lysed usingHNET buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EGTA, and 1% TritonX-100) supplemented with 1 mM DTT, 1 mM PMSF and protease inhibitorscocktail (Boehringer Mannheim, Germany): 35 μg of protein was loaded on10% SDS-PAGE followed by transferring to PVDF membrane. Immunoblottingwas performed as previously described using a p53 mouse monoclonalantibody from Oncogene Research Products (Lee et al. (1999) J Biol Chem274:13451-5).

[0123] To measure p53 half-life cycloheximide (30 μg/ml) was added toMCF-7 cells 30 min after the addition of triptolide and harvested at thetimes shown for immunoblot analysis of p53. Immunoblot analysis usingother antibodies was performed as described above. The band intensitywas measured by NIH Image 1.62.

[0124] Sub-cellular fractionation of MCF-7 cells. After treatment withtriptolide (5 or 20 ng/ml) and/or doxorubicin (100 nM) cytosolic andnuclear extracts were prepared as previously described (Lee et al.(1988) Gene Anal Tech 5:22-31) and 50 μg of each extract was used inSDS/PAGE immunoblot analysis of p53.

[0125] Metabolic labeling of MCF-7 cells. Cells were grown to 80%confluence followed by pretreatment with triptolide (20 ng/ml) for 6 hin the appropriate medium. Cells were washed twice with short-termlabeling medium (RPMI with 5% dialyzed FCS supplemented withL-glutamine, penicillin, and streptomycin). To deplete intracellularpools of methionine short-term labeling medium was added for 15 min at37° C., then replaced by short-term labeling medium containing 0.1mCi/ml [³⁵S] methionine (Amersham, Inc.). Cells were labeled for 30 minat 37° C. and washed with ice-cold PBS before harvesting forimmunoprecipitation. The cells were lysed using RIPA buffer supplementedwith protease inhibitors and immunoprecipitated using anagarose-conjugated p53 mAb (Ab-6, Oncogene Research Products) followedby 10% SDS-PAGE. The intensity of labeled p53 protein was measured byNIH Image 1.62.

[0126] Results

[0127] Triptolide induces apoptosis in solid tumor cell lines andenhances chemotherapy-induced apoptosis. To determine if tumor celllines are sensitized to chemotherapeutic agents in the presence oftriptolide, a topoisomerase II inhibitor, doxorubicin, was used.Doxorubicin (100 nM alone in A549 and HT1080 cells caused only a slightdecrease in cell viability, 14.3 and 6.4% respectively, after 48 hoursof drug treatment (Table 1). However, in HT-1080 cells, the combinationof triptolide at 5 ng/ml (13.5 nM) plus doxorubicin reduced cellviability by 65%, but triptolide at 5 ng/ml or doxorubicin (100 nM)alone reduced cell viability only by 10% and 6% respectively. Triptolideat 20 ng/ml (54 nM) alone reduced cell viability by 74% in HT1080 cells.Also, in A549 cells, the combination of triptolide at 20 ng/ml plusdoxorubicin (100 nM) decreased cell viability by 67% but triptolide anddoxorubicin alone decreased viability only by 35% and 15% respectively.

[0128] Additionally, we observed that triptolide enhances cell death inA549 cells induced by carboplatinum, another topoisomerase II inhibitor.We also examined the effect of triptolide (20 ng/ml) alone on the MCF-7breast cancer cell line which contains wild-type p53. We found thattriptolide, 5 ng/ml and 20 ng/ml, decreased cell viability by 36% and70% respectively in MCF-7 cells (Table 2). We have also found thattriptolide alone induces cell death in greater than 80% of cells inother solid tumor cell lines. Thus, triptolide alone is cytotoxic intumor cells and it cooperates with doxorubicin to enhance cell death intumor cell lines. TABLE 2 Cell viability assay of human tumor cell linesafter triptolide treatment Percent survival^(a) Treatment MCF-7 A549HT-1080 Triptolide 5 ng/ml 63.9 ± 8.1 91.1 ± 3.8  90.4 ± 6.2  Triptolide20 ng/ml 30.5 ± 7.6 64.0 ± 8.2  26.0 ± 5.2  Doxorubicin 100 nM ND^(b)85.7 ± 9.6  93.6 ± 4.3  Triptolide 5 ng/ml + ND  76.5 ± 9.9  35.8 ± 6.7 Doxorubicin 100 nM Triptolide 20 ng/ml + ND  33.6 ± 11.4 15.5 ± 1.4 Doxorubicin 100 nM

[0129] Triptolide increases expression of p53. p53 mediates cell deathresponses to cytotoxic stimuli such as hypoxia, irradiation and DNAdamaging chemotherapeutic agents. Since triptolide alone is cytotoxicand it cooperates with doxorubicin, it was hypothesized thattriptolide-induced apoptosis may be mediated by p53. In both MCF-7 andA549 cells, which retain wild-type p53, triptolide increased p53 steadystate protein levels 2-4 fold in a dose- and time-dependent manner. InMCF-7 cells doxorubicin induced a 2 fold increase in p53, and triptolideinduced a greater than 4-fold increase in p53 protein. In A549 cells,the combination of triptolide (20 ng/ml) plus doxorubicin (100 nM) at 24h showed the greatest increase (greater than a 12-fold increase) in p53.Triptolide (5 ng/ml) in combination with doxorubicin also markedlyincreased p53 in HT1080 cells. We next examined if the increase in thep53 protein level was due to an increase in the p53 mRNA. The levels ofthe p53 mRNA did not increase in response to triptolide but, in fact,p53 mRNA was slightly reduced in MCF-7 cells treated for 16 h withtriptolide (FIG. 5A).

[0130] In the experiments shown in FIG. 5, RT-PCR was performed using 2μg of total RNAs extracted from MCF-7 cells. Cells were treated withtriptolide (20 ng/ml) or doxorubicin (100 nM) and harvested after 8 and16 hours. GADPH was used as a loading control. The plasmid pBP100-GL2which contains a p53-binding site in the mdm2 promoter was transientlytransfected into MCF-7 cells, and cellular lysates were used for theluciferase assay. The values are an average of three experiments±S.D.Taken together, these data suggest that the increase in p53 ispost-transcriptional in cells undergoing triptolide-induced cell death.

[0131] Functional p53 enhances triptolide-induced cell death. Theoutcome of many chemotherapeutic drugs or radiation therapy depends onthe functional status of the tumor suppressor p53 gene. To determine ifthe presence of functional p53 contributes to triptolide-induced celldeath, we used mouse embryonic fibroblasts (MEFs) cells with thewild-type (+/+) or null (−/−) p53 gene. Triptolide at dosages of 5 ng/mlor 10 ng/ml reduced p53+/+ MEF cell viability by 48% and 73%respectively and by 15% and 50% in p53 (−/−) cells (Table 3). In MEFcells with the wild-type p53, doxorubicin induced 35% more cell deaththan those without functional p53. Also, the combination of triptolideplus doxorubicin reduced cell viability by 88% in p53 (+/+) cells butonly by 55% in p53 (−/−) cells. Therefore, functional p53 plays a rolein mediating triptolide-induced cell death.

[0132] Expression of Mdm2 and p21 are down-regulated in cells treatedwith triptolide. One model of p53-mediated apoptosis is that uponcellular stresses (such as DNA damage), p53 is stabilized and thisincreases expression of genes such as mdm2, bax, p21^(Cip1/Waf1) andgadd45. Mdm2 negatively regulates p53 stability by mediating nuclearexport via direct protein binding and/or ubiquitin/proteosomedegradation. In DNA damage (such as γ-irradiation), phosphorylations ofp53 on serines 15 and 392 by DNA-PK or ATM interferes with the abilityof Mdm2 to bind to p53 and target p53 for degradation. This results instabilization and activation of p53.

[0133] To determine if a similar mechanism exists in triptolide-inducedapoptosis, the levels of several genes that are downstream of p53transactivation were examined. When MCF-7 cells were treated withdoxorubicin 100 nM, there was about a 1.5-2 fold increase in the Mdm2mRNA and protein. This increase in Mdm2 paralleled the increase in p53level which also resulted in increases in bax and p21 mRNA.

[0134] In cells treated with triptolide, however, there was atime-dependent decrease in mdm2 mRNA. To measure the effect oftriptolide on mdm2 gene expression, a luciferase vector was used, whichcontains a consensus p53-binding site from the mdm2 promoter. Despitethe high levels of p53 in triptolide-treated MCF-7 cells,transactivation of the reporter construct decreased by approximately 30%in the presence of triptolide. However, doxorubicin increasedtransactivation of the Mdm2 by 15% by 16 h. The repression of the p53dependent genes by triptolide is not a general effect, since gadd45 andelongation factor 1-alpha (EF-1α), which are also induced by p53, werenot affected. Thus, triptolide induces p53 but represses expression ofsome p53 dependent genes.

[0135] To determine if the absence of an increase in p53 target genes incells treated with triptolide is due to the lack of p53 translocation,p53 translocation into the nucleus was examined after triptolidetreatment. Compared with the cells treated with doxorubicin, where themajority of p53 is translocated into the nuclei, the majority of p53 incells treated with triptolide (20.ng/ml) was also translocated intonuclei.

[0136] There was no significant change in the levels of the Mdm2 proteinin MCF-7 cells treated with 5 ng/ml of triptolide for 8 or 24 hours buttriptolide reduced cell viability by only 10% at this dosage. There wasan approximately 1.5-fold increase in Mdm2 in MCF-7 cells treated with20 ng/ml of triptolide at 8 h but by 24 h there was almost a completeloss of Mdm2 protein (FIG. 4). Also, there was a 3-fold decrease in thelevel of p21 protein in triptolide-treated MCF-7 cells but nosignificant change in Bax.

[0137] Triptolide induces translation of p53. To determine the mechanismby which triptolide induces p53 we examined the effect of triptolide onp53 protein stability and translation. To examine the effect onstability we examined levels of p53 in the presence of cycloheximide (30μg/ml) in MCF-7 cells, a dose which blocks translation. When cells werepretreated with triptolide for 0.5 h prior to the addition ofcycloheximide, there was a slight increase in p53 stability at 30 minbut there was no difference from untreated cells at 60 min. These datasuggested that the increased steady-state level of the p53 protein inresponse to triptolide did not result from an increase in the half-lifeof the p53 protein. We then examined if triptolide induces translationof p53 by in vivo [³⁵S]methionine metabolic labeling of MCF-7 cells. Wefound, interestingly, that triptolide induced a 4.9-fold increase in p53translation (FIG. 5B). Thus, triptolide-induced p53 accumulation ismediated by an increase in p53 translation.

[0138] Triptolide induces cell death in almost 70% of MCF-7 cells andenhances chemotherapy-induced cell death in A549 and HT1080 cells. Todelineate possible mechanism(s) of triptolide-mediated apoptosis, therole of the p53 tumor suppressor gene was studied. Triptolide inducedp53 protein expression in several wild-type p53 tumor cell lines andwild-type p53 significantly enhanced the cytotoxicity of triptolide.Interestingly, triptolide induced cell death in over 80% of cells in amutant p53 lung cancer cell line so that functional p53 is not requiredfor triptolide-induced apoptosis. The data presented here suggests thattriptolide alone and in combination with DNA damaging agents mediates ap53-dependent dependent apoptotic pathway in tumor cells with wild-typep53. It was observed that triptolide increased levels of p53 at apost-transcriptional level. This was mediated by a 5-fold increase inp53.

[0139] A late decrease in Mdm2 protein in triptolide-treated cellsprovide an additional mechanism for the increase in p53, and a possiblemechanism for how triptolide sustains induction of p53 in the presenceof DNA-damaging agents. Triptolide-mediated repression of downstream p53genes may serve to inhibit expression of survival factors such as MAP4and the IGF1 receptor. Since triptolide shows enhanced cytotoxicity incombination with DNA damaging agents, it may also interfere with DNArepair. Triptolide, however, does not induce DNA strand breaks asrevealed by a comet assay.

[0140] The above results demonstrate that triptolide induces p53 andthat functional p53 enhances triptolide-induced apoptosis. It is alsoshown that triptolide enhances the cytotoxicity of DNA damaging agents.The cytotoxic activity of triptolide alone and its ability to cooperatewith other cytotoxic agents represents a novel method to enhancecytolysis of solid tumor cells in vivo.

Example 3 Synergistic Combination with CPT-11

[0141] In an animal model, it was shown that the combination of CPT-11and PG490-88 provided for synergistic killing of tumor cells.

[0142] Materials and Methods

[0143] Mice. Female NCr nude mice were purchased from Taconic,Germantown, N.Y., and were generally 20-24 grams when used. Mice werekept in autoclaved filter-top microisolator cages with autoclaved waterand sterile food ad lib. The cages were maintained in an isolator unitproviding filtered air (Lab Products, Inc., Maywood, N.J.).

[0144] Nude mouse xenograft model. HT1080 tumor cells were grown intissue culture flasks and harvested using EDTA and trypsin. Cells werecentrifuged and the concentration of viable cells was appropriatelyadjusted. Female NCr nude mice were injected intradermally with 5million HT1080 tumor cells each. Tumor size was monitored after tumorcell implantation by measuring the width, length and thickness of thetumors and using a formula to calculate the volume. When an appropriatetumor volume was achieved, the mice were grouped together to constitutea similar mean tumor size in each group in the experiment, and treatmentwas initiated. Control mice were left untreated. PG490-88 treated micereceived IP injections of PG490-88 in phosphate buffered saline (0.75mg/kg) on days 0-5 and 7-11. CPT-11 treated mice were given IVinjections with CPT-11 in phosphate buffered saline (11 mg/kg) on days1, 5, and 9. Combination therapy mice received both PG490-88 and CPT-11treatments.

[0145] The results are shown in FIG. 6, and Table 3. TABLE 3 Day 0 Day 4Day 7 Day 10 Day 14 Summary TV Mean SE TV Mean SE TV Mean SE TV Mean SETV Mean SE Control 103 15 293 49 536 69 616 164 980 380 PH490-88 0.75 IP5X/wk 107 20 167 14 208 18 247 21 532 55 CPT-11 11 IV every 4 days 10736 157 60 209 65 268 72 495 112 PG490-88 (0.75) IP + CPT-11 (11) IV 10731 106 22 35 7 12 3 1 0

Example 4 PG490-88 Used in Combination with Navelbine

[0146] PG490-88 exerts anticancer effects upon tumors established usingthe human tumor xenograft model in nude mice using cells from humantumor cell lines. Navelbine is a chemotherapeutic agent derivedsemisynthetically from vinblastine that is being used more widely,partly because of a milder side effect profile. PG490-88 was tested incombination with Navelbine in nude mice bearing established HT-29 coloncancer tumors.

[0147] HT-29 tumor cells grown in tissue culture were harvested andimplanted intradermally on the backs of nude mice. Tumor size wasmonitored by measuring the dimensions of each tumor and calculatingtumor volume with a formula. When the tumors reached an appropriatesize, the mice were grouped together (5 mice/group) to constitute asimilar mean tumor size (approximately 100 mm³), and treatment wasinitiated on day 0. Vehicle-treated mice received 0.9% NaCl i.p. dailyfor 5 days per week for 2 weeks. PG490-88 treatment at 0.25 mg/kg wasadministered daily i.p. 5 days per week for 2 weeks. Navelbine at 10mg/kg was given i.v. on days 0 and 7. Mice in the combination treatmentgroup received PG490-88 i.p. and Navelbine i.v. at the appropriatetimes. The treatments were given to mice at 100 μl per 10 g of mousebody weight. Tumor dimensions were measured periodically for calculationof tumor volumes.

[0148]FIG. 7 shows the change in tumor volume with treatment. The dataare presented as mean percent change in tumor volume from day 0 for eachtreatment group. Day 0 tumor volumes are shown in Table 4. Toxicity ofthe treatment was monitored by weighing the mice daily 5 days per week.

[0149] In FIG. 8, a comparison is provided for tumor volumes on Days 0and 14. Tumor dimensions were measured periodically for calculation oftumor volumes, and the final determinations were made on day 14. Thedata are presented as mean tumor volume in mm³ for each treatment groupfor the measurements on days 0 and 14.

[0150] PG490-88 at 0.25 mg/kg exerted a modest effect upon HT-29 tumorvolume, with an increase by day 14 of 238% compared to the vehiclecontrol increment of 372% from day 0, FIGS. 7 and 8, Table 4). Navelbinehad little impact upon growth of HT-29 tumors, producing a 343% increasein tumor volume. Although there was little effect upon tumor growth byeither agent alone, PG490-88 and Navelbine caused tumor regression whenused in combination therapy. The drug combination produced a 29%reduction in mean tumor volume by day 14, and the regression was evidentby day 10. As measured on day 14, the tumors on 0.4 of 5 of the mice inthe combination therapy group had decreased in size compared to day 0.TABLE 4 PG490-88 treatment of nude mice bearing established HT-29 humancolon cancer cell line tumors. Day 0 Tumor Volume (mm³) Treatment GroupMean S.E. Saline 108 6 PG490-88 0.25 mg/kg 109 7 Navelbine 10 mg/kg 1096 PG490-88 0.25 mg/kg + Navelbine 108 7 10 mg/kg

[0151] Nude mice with established HT-29 tumors were grouped together toconstitute a similar mean tumor size in each group, and the mean tumorvolumes and standard errors (SE) on day 0 are presented.

Example 5 Role of p21

[0152] Materials and Methods

[0153] Reagents. PG490 (triptolide, MW 360) was obtained fromPharmagenesis (Palo Alto, Calif.). A549 (non-small cell lung cancer) andHT1080 (fibrosarcoma) cell lines were from ATCC. Mouse embryonicfibroblasts (p53+/+ and p53[−]/[−]) cell lines were provided by Dr.Amato J. Giaccia (Stanford University). Doxorubicin, cycloheximide, andthe anti-FLAG (M2) antibody were obtained from Sigma. Antibodies forp53, p21waf1/cip1 and protein phosphatase-1 were from Calbiochem.

[0154] Cell Culture and Plasmids. A549 and HT1080 cells were cultured inthe appropriate media with 10% fetal calf serum supplemented withL-glutamine, penicillin, and streptomycin. p53 wild-type (+/+) and null([−]/[−]) mouse embryonic fibroblasts (MEFs) transfected with theE1A/Ras were grown in Dulbecco's modified Eagle's medium containing 15%fetal calf serum supplemented with L-glutamine, penicillin, andstreptomycin.

[0155] The full-length coding region of human p21 cDNA was amplified byRT-PCR from HT1080 cells with oligo primers 5′-(SEQ ID NO:9)GGATCCGCCACCATGTCAGAACCGGCTGGGG-3′ and 5′-(SEQ ID NO:10)GTCGACTCACTTGTCATCGTCGTCCTTGTAGTCCTCGAGGGGCTTCCTCTTGGAGAAGA TCAG-3′. The3′ primer was manipulated to add an in-frame FLAG-tag sequence beforethe stop codon. Subsequently, the cDNA fragments were cloned into thepIND, ecdysone-inducible vector (Invitrogen, Carlsbad, Calif.). Thefull-length sequence of human p21 coding region was confirmed by DNAsequencing. The cDNA of [beta]-galactosidase (LacZ) was cloned into thesame vector as a control. Transfection into HT1080 cells was performedwith the LipofectAMINE Plus kit (Life Technologies, Inc.) according tothe manufacturers protocol. Briefly, HT-1080 cells at 70% confluencewere cotransfected with 1 μg of pINDp21FLAG or pINDLacZ-FLAG plus 1 μgof pVgRXR (Invitrogen, Carsbad, Calif.) in 6-well plates. Afterincubation for 3 h at 37° C., the medium was replaced with fresh media.Then 48 h after transfection, cells from each well were transferred intothree 10-cm culture dishes. After overnight culture, stabletransfectants were selected by adding 600 μg/ml of zeocin (Invitrogen)and 800 μg/ml G-418 (Life Technologies). The selection was carried onfor 2 weeks. Individual clones were isolated and tested for proteinexpression induced by the addition of 5 μM ponasterone A (Invitrogen).

[0156] Cell Viability Assay. HT-0.1080 cells were seeded into 6-wellplates at 2×10⁵ per well the day before the treatment. For the inducibleexpression of exogenous p21 and LacZ, the stable transfectants werecultured in the presence of 5 μM ponasterone A for 16 h. Three fieldsfrom each well were carefully selected, marked, and counted to ensure asimilar cell number greater than 300 cells per field before thetreatment. The cells were untreated or treated with doxorubicin,triptolide, or the combination of both at indicated dosages for 8 h at37° C. Subsequently, the medium was replaced with new media plus 5 μMponasterone A. After 16 h incubation at 37° C., the number of viablecells within the same fields were determined by trypan blue exclusionwith a 2% trypan blue solution. Cell death was confirmed as apoptotic byannexin V/propidium iodide (PI) staining followed by FACS analysis.

[0157] Northern Blot Analysis. RNA was prepared from HT1080 cells usingRNeasy Mini Kit from Qiagen Inc. (Valencia, Calif.). cDNAs for Northernblot analysis for p21 and p53 were prepared using RT-PCR with 2 μg oftotal RNA. The following primer pairs were used: p53, (SEQ ID NO:11)5′-AGTCAGATCCTAGCGTCGAG-3′ and (SEQ ID NO:12)5′-TCTTCTTTGGCTGGGGAGAG-3′; p21, (SEQ ID NO:13)5′-AGTGGGGCATCATCAAAAAC-3′ and (SEQ ID NO:14)5′-GACTCCTTGTTCCGCTGCTAATC-3′; and glyceraldehyde-3-phosphatedehydrogenase, (SEQ ID NO:15) 5′-CCCATCACCATCTTCCAG-3′ and 5′-(SEQ IDNO:16) ATGACCTTGCCCACAGCC-3′. Northern blot analysis was performed asdescribed previously (Lee et al. (1999) J. Biol. Chem. 274.13451-13455).

[0158] Electromobility Shift Assay (EMSA. HT1080 cells were treated asshown. The EMSA was performed as described previously using anend-labeled 32P-p53 consensus binding site (Santa Cruz Biotechnology,Santa Cruz, Calif.) (Lee et al., supra.)

[0159] Immunoblotting. Cells were harvested at the conditions and timesindicated and lysed using HNET buffer (50 mM HEPES, pH 7.5, 100 mM NaCl,1 mM EGTA, and 1% Triton X-100) supplemented with 1 mM dithiothreitol, 1mM phenylmethylsulfonyl fluoride, and protease inhibitors mixture (RocheMolecular Biochemicals). 100 μg of protein was loaded on 10% SDS-PAGEfollowed by transferring to polyvinylidene difluoride membrane.Immunoblotting was performed as described previously using a p53 mousemonoclonal antibody from Oncogene Research Products. To measure p53half-life, cycloheximide (30 μg/ml) was added to HT1080 cells 30 minafter the addition of triptolide and harvested at the times shown forimmunoblot analysis of p53. Immunoblot analysis using other antibodieswas performed as described above. The band intensity was measured by NIHImage 1.62.

[0160] Cell Cycle Analysis. HT1080 cells (2×10⁶) were treated withtriptolide (20 ng/ml) and/or doxorubicin (100 nM) for 16 h. Cells werethen harvested and washed with cold PBS. The cells were resuspendedgently in 5 ml of 100% ethanol and fixed at 25° C. for 1 h. Afterwashing with PBS, the cells were incubated with DNase-free RNase A (200μg/ml) at 37° C. for 1 h and washed with PBS. Propidium iodide (10μg/ml) was added, and the cells were incubated at 37° C. for 5 min.Cells were separated by sonicating at 20% output level for 15 s using aVirSonic 50 sonicator (Vitis Inc., NY). The samples were then sorted byFACS, and cell cycle analysis was done with FlowJo (version 3.0.3) (TreeStart, Inc, San Carlos, Calif.).

[0161] Metabolic Labeling of HT1080 Cells. Cells were grown to 80%confluence followed by pretreatment with triptolide (20 ng/ml) for 6 hin the appropriate medium. Cells were washed twice with short termlabeling medium (RPMI with 5% dialyzed fetal calf serum supplementedwith L-glutamine, penicillin, and streptomycin). To depleteintracellular pools of methionine, short term labeling medium was addedfor 15 min at 37° C. and then replaced by short term labeling mediumcontaining 0.1 mCi/ml [35S]methionine (Amersham Pharmacia Biotech).Cells were labeled-for 30 min at 37° C. and washed with ice-cold PBSbefore harvesting for immunoprecipitation. The cells were lysed usingRIPA buffer supplemented with protease inhibitors and immunoprecipitatedusing an agarose-conjugated p53 mAb (Ab-6, Oncogene Research Products)followed by 10% SDS-PAGE. The intensity of labeled p53 protein wasmeasured by NIH Image 1.62.

[0162] In Vivo [³²P]Orthophosphate Labeling. Subconfluent HT1080 cellswere pretreated with 20 ng/ml triptolide for 2 h in Dulbecco's modifiedEagle's medium supplemented with 10% fetal bovine serum and antibiotics.These cells were washed twice with 37° C. labeling medium (Dulbecco'smodified Eagle's medium, 10% fetal bovine serum dialyzed againstphosphate-free) lacking sodium phosphate. Cells were then labeled for anadditional 1 h with the labeling medium containing 1 mCi/ml[32P]orthophosphate and 20 ng/ml triptolide. The labeling medium wasremoved, and the cells were washed three times with cold Tris-bufferedsaline (TBS). The cells were scraped in 1 ml of lysis buffer (1% NonidetP-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodiumphosphate, pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodiumvanadate, and 100 units/ml aprotinin) and kept on ice for 10 min.Lysates were passed through 27-gauge needles and spun at 11,000×g for 10min, and 100 μg of the supernatants were subjected toimmunoprecipitation with p53 mAb (Ab-6) conjugated with agarose for 4 h.Samples were then spun at 2500 rpm at 4° C. for 5 min and washed threetimes with the lysis buffer. The pellets were boiled in SDS samplebuffer and analyzed on 10% SDS-PAGE. The bands were visualized andquantified by Optiquant Phosphorimager.

[0163] Results

[0164] Triptolide Enhances Chemotherapy-induced Apoptosis. After 24 h oftreatment, doxorubicin (100 nM) or triptolide (5 ng/ml) alone did notreduce HT1080 cell viability. The combination of triptolide (5 ng/ml)plus doxorubicin reduced cell viability by 65%. Triptolide alone at 20ng/ml reduced HT1080 cell viability by 84%. Cytotoxic synergy betweentriptolide and doxorubicin was also observed in A549 lung cancer cells,and triptolide also enhanced cell death by carboplatinum, anothertopoisomerase II inhibitor, in A549 and HT1080 cells. Doxorubicin didnot induce NF-κB transcriptional activity in HT1080 cells so thattriptolide is not enhancing doxorubicin-mediated apoptosis throughinhibition of NF-κB:

[0165] Triptolide Induces p53 but Inhibits p21 Expression. p53 mediatescell death responses to cytotoxic stimuli such as hypoxia, irradiation,and DNA-damaging chemotherapeutic agents. Since triptolide alone iscytotoxic and it cooperates with DNA-damaging chemotherapeutic agents,we hypothesized that triptolide-induced apoptosis may be mediated byp53. In HT1080 cells that contain wild-type p53, triptolide (20 ng/ml)increased p53 steady-state protein levels 4-fold for 9 h, and triptolide(5 ng/ml) induced a 2.4-fold increase in p53. Doxorubicin induced a4.9-fold increase in p53, and the combination of doxorubicin plustriptolide induced a 4-fold increase in p53 protein. In A549 cells, thecombination of triptolide (20 ng/ml) plus doxorubicin (100 nM) at 24 hshowed greater than 5-fold increase in p53. We next examined if theincrease in the p53 protein level was due to an increase in the p53mRNA. The level of p53 mRNA was not affected by triptolide. These datasuggest, therefore, that triptolide induces post-transcriptionalaccumulation of p53.

[0166] A current model of p53-mediated apoptosis is that upon cellularstresses (such as DNA damage), p53 is stabilized, and this increasesexpression of genes such as mdm2, bax, p21cip1/waf1, and gadd45. Recentstudies show that doxorubicin and γ-irradiation-mediated activation ofp53 in p53 wild-type cells induces p21 and causes growth arrest whichinhibits apoptosis. To determine whether triptolide enhancesdoxorubicin-mediated apoptosis by blocking p21-mediated growth arrest,we examined the effect of triptolide on p21 expression. Triptolide (20ng/ml) reduced basal p21 levels by 50% despite inducing p53. Doxorubicininduced a 14.5-fold increase in p21 that was completely blocked bytriptolide (20 ng/ml), and triptolide (5 ng/ml) reduceddoxorubicin-mediated induction of p53 by 58%.

[0167] Triptolide Inhibits p21 mRNA Expression. Triptolide (20 ng/ml)also completely blocked doxorubicin-mediated induction of p21 mRNA (FIG.9A). Triptolide or doxorubicin did not affect p21 expression, in the p53mutant HT29 colon cancer cell line. Triptolide also blockeddoxorubicin-mediated induction of mdm2 mRNA, but it did not affectgadd45 or map4 mRNA expression (FIG. 9B). These data show thattriptolide alone inhibits transcription of p21, and it blocksdoxorubicin-mediated transcriptional induction of p21 despite increasingp53 levels.

[0168] Triptolide does not inhibit DNA binding of p53. Triptolideinduces p53 but inhibits p21 expression. Also, triptolide blocksdoxorubicin-mediated induction of p21. We then performed EMSA todetermine whether triptolide, alone or in combination with doxorubicin,inhibits DNA binding of p53 to a p53 consensus binding site in the p21promoter. Triptolide alone slightly enhanced DNA binding of p53, and itdid not block doxorubicin-mediated induction of DNA binding. These datasuggest triptolide represses expression of p21 by blockingtransactivation but not DNA binding of p53.

[0169] Triptolide Inhibits p21-mediated Growth Arrest. We then evaluatedthe effect of triptolide alone and in combination with chemotherapy oncell cycle progression. Triptolide (20 ng/ml) alone increased the numberof cells in S phase from 23.7% in unstimulated cells to 46% intriptolide-treated cells. Doxorubicin induced accumulation of cells inG2/M from 11 to 49.8%, but triptolide inhibited doxorubicin-mediatedG2/M accumulation from 49.8 to 22.6%. Since p21 mediates G2/M arrest inp53 wild-type cells in response to chemotherapy, our data suggest thattriptolide inhibits doxorubicin-mediated G2/M arrest by blockinginduction of p21.

[0170] Overexpression of p21 Inhibits Cytotoxic Synergy betweenTriptolide and Doxorubicin. To determine whether triptolide-mediatedinhibition of p21 is involved in the cytotoxic synergy betweentriptolide and doxorubicin, we overexpressed p21 in HT1080 cells using aponasterone-inducible p21 vector (pIND-p21). The addition of ponasteroneA (5 μM) strongly induced exogenous p21 expression that was slightlyreduced by triptolide plus doxorubicin. The combination of triptolide (5ng/ml) and doxorubicin (100 nM) reduced cell viability to 30% in theinduced vector control, and viability increased to 58% following theinduction of exogenous p21.

[0171] Triptolide Induces Translation of p53. To determine the mechanismby which triptolide induces p53, we examined the effect of triptolide onp53 protein stability and translation. To examine the effect onstability, we examined levels of p53 in the presence of cycloheximide(30 μg/ml) in. HT1080 cells, a dose that blocks translation. In cellsthat were pretreated with triptolide (20 ng/ml) for 0.5 h prior to theaddition of cycloheximide, there was a slight increase in p53 stabilityat 30 min, but there was no difference from untreated cells at 60 min.These data suggested that the increased steady-state level of the p53protein in response to triptolide did not result from an increase in thehalf-life of the p53 protein. We then examined if triptolide inducestranslation of p53 by in vivo ³⁵S methionine metabolic labeling ofHT1080 cells. We found, interestingly, that triptolide induced a4.9-fold increase in p53 translation. Thus, triptolide-induced p53accumulation is mediated by an increase in p53 translation.

[0172] Triptolide Induces Phosphorylation of p53. Many studies haveshown that phosphorylation of p53 in response to DNA damage regulatesp53-mediated apoptosis and transcriptional activity. Therefore, weexamined if p53 undergoes hyperphosphorylation upon triptolide treatmentin HT1080 cells. There was a 2-3-fold increase in phosphorylated p53 intriptolide-treated cells compared with p53 from the untreated cells.Western blot analyses of the samples show equivalent levels ofimmunoprecipitated p53 in both samples. These data show that triptolideinduces phosphorylation of p53 at 3 h that is prior to thetriptolide-mediated induction of p53 protein expression. Synergy betweentriptolide and CPT-11 in vitro, in HT-1080 cells. PG490 (5 CPT-11 CPT-11PG490 + PG490 + CPT- Treatment Control ng/ml) (5 M) (10 M) CPT-11 (5 μM)11 (10 μM) Apoptotic 0% 13.5% 0% 5% 23% 69% Cells (%)

[0173] HT1080 cells were treated as shown and harvested for analysis ofapoptosis by Annexin V and Propidium Iodide staining 20 h aftertreatment. In experiments with a combination of PG490 and CPT-11, cellswere pretreated with PG490 for 4 h followed by the addition of CPT-11for 16 h. It was also found that PG490 (triptolide) inhibitsCPT-11-mediated induction of p21 in HT1080 and A549 cells.

[0174] These above data demonstrate that triptolide enhanceschemotherapy-induced cell death in p53 wild-type cells. Triptolideinduced p53 protein expression but inhibits basal p21 expression anddoxorubicin-mediated induction of p21. We observed that p21 levels arealmost undetectable in p53 cells suggesting that p53 is also requiredfor basal p21. These data likely reflect the inhibitory effect oftriptolide on transcriptional activity but not DNA binding of p53 whichis analogous to triptolide blocking transactivation and not DNA bindingof NF-κB. Triptolide, however, is not a general transcriptionalinhibitor because it does not inhibit growth arrest and DNAdamage-inducible (gadd45) elongation factor-α (EF-1α) orglyceraldehyde-3-phosphate dehydrogenase expression (GAPDH).Additionally, we observed that triptolide induces phosphorylation ofp53, but this is the first example of a modification of p53 thatinhibits p21 expression.

[0175] Triptolide inhibits p21-mediated accumulation of cells in G2/Mand induces accumulation of cells in S phase. Also, in combination withdoxorubicin, triptolide enhances apoptosis in p53 wild-type tumor cells,and overexpression of p21 inhibits the cytotoxic synergy betweentriptolide and doxorubicin. Triptolide, however, does not repress p21expression in p53 mutant tumor cells, but it does induce accumulation ofp53 mutant cells in S phase. Triptolide may directly inhibit cyclinsrequired for G1/S transition such as cyclin A, cyclin D, and cyclin E orblock cdk-4 or cdk-6 activity. We show here that not only do triptolideand doxorubicin affect different cell cycle checkpoints but triptolideblocks p21-mediated G2/M arrest which likely enhances apoptosis byblocking growth arrest. The data suggest that triptolide enhancesdoxorubicin-mediated apoptosis, by blocking p21-mediated G2/M arrest.

[0176] Since triptolide shows enhanced cytotoxicity in combination withDNA-damaging agents, it may also interfere with DNA repair. It has beenreported that casein kinase II phosphorylates p53 at serine 386 whichcauses p53-mediated repression. The proline-rich region of the p53protein has been shown to be important in chromatin remodeling and isrequired to overcome p53-mediated transcriptional repression. Thecytotoxic activity of triptolide alone and its ability to cooperate withother cytotoxic agents provides a novel method to enhance cytolysis oftumor cells in vivo. In support of this observation, we have found thatPG490-88, a water-soluble derivative of triptolide, cooperates withchemotherapy to cause tumor regression in a tumor xenograft model.

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What is claimed is:
 1. A method for treatment of a hyperproliferativedisorder, the method comprising: contacting a targeted cell populationwith a combination of an anti-proliferative agent; and a diterpenoidtriepoxide having the structure:

wherein X₁ is OH, ═O; or OR¹; X₂ and X₃ are independently OH, OR¹ or H;R¹ is —C(O)—Y-Z, wherein Y is a branched or unbranched C₁ to C₆ alkyl oralkenyl group; and Z is COOR², NR³R^(3′), or +NR⁴R^(4′), R^(4″), whereR² is a cation; R³ and R^(3′) are independently H or branched orunbranched C₁ to C₆ alkyl, hydroxyalkyl, or alkoxyalkyl, or R³ andR^(3′) taken together form a 5- to 7-member heterocyclic ring whose ringatoms are selected from the group consisting of carbon, nitrogen, oxygenand sulfur, wherein the ring atoms include 2 to 6 carbon atoms, or morenitrogen atoms, and optionally one or more oxygen or sulfur atoms, andwherein the ring is unsubstituted or is substituted with one or moregroups selected from R⁵, OR⁵, NR⁵R⁶, SR⁵, NO₂, CN, C(O)R⁵, C(O)NR⁵R⁶,and halogen (fluoro, chloro, bromo, or iodo), where R⁵ and R⁶ areindependently hydrogen, lower alkyl or lower alkenyl; and R⁴, R^(4′) andR^(4″) are independently branched or unbranched C, to C₆ alkyl,hydroxyalkyl or alkoxyalkyl; in a combined dosage effective tosubstantially reduce the numbers of said targeted cell population. 2.The method of claim 1, wherein said diterpenoid triepoxide is selectedfrom the group consisting of triptolide, tripdiolide,16-hydroxytriptolide; triptolide succinate, an ester derivative of,triptolide, an ester derivative of tripdiolide, and an ester derivativeof 16-hydroxytriptolide.
 3. The method of claim 1, wherein saidhyperproliferative disorder is selected from the group consisting ofcancer, restenosis, atherosclerosis, in-stent stenosis, vascular graftrestenosis, fibrotic disorders, psoriasis, inflammatory disorders,arthritis, glomerular nephritis, endometriosis, macular degenerativedisorders, and autoimmune disorders.
 4. The method of claim 1, whereinsaid hyperproliferative disorder is a cancer.
 5. The method of claim 1,wherein said tumor is a human tumor.
 6. The method of claim 5, whereinsaid tumor is a solid tumor.
 7. The method of claim 6, wherein saidtumor is a carcinoma.
 8. The method of claim 4, wherein said cancer ismulti-drug resistant.
 9. The method of claim 4, wherein said cancerexpresses functional p53 protein.
 10. The method of claim 4, whereinsaid cancer expresses p21^(waf1/cip1).
 11. The method according to claim10, wherein said expression of p21^(waf1/cip1) is upregulated inresponse to said anti-proliferative agent.
 12. The method of claim 4,wherein said anti-proliferative agent is a topoisomerase inhibitor. 13.The method of claim 12, wherein said topoisomerase inhibitor is ananthracycline.
 14. The method according to claim 13, wherein saidanthracycline is selected from the group consisting of doxorubicin,daunorubicin, epirubicin, idarubicin, anamycin, and MEN
 10755. 15. Themethod of claim 1, wherein said diterpenoid triepoxide and saidanti-proliferative agent are administered in a co-formulation.
 16. Themethod of claim 1, wherein said diterpenoid triepoxide and saidanti-proliferative agent are separately formulated.
 17. The methodaccording to claim 1, wherein said combination of an anti-proliferativeagent; and a diterpenoid triepoxide provide for a synergistic response.18. The method of claim 1, wherein said anti-proliferative agent is aDNA-damaging compound.
 19. The method of claim 17, wherein saidanti-proliferative agent is taxol.
 20. A method of screening a tumor forbenefit from a combination therapy comprising an anti-proliferativeagent and a diterpenoid triepoxide, the method comprising: obtaining asample of cells from said tumor; determining if said cells expressp21^(waf1/cip1); wherein tumors comprising cells that express saidp21^(waf1/cip1) benefit from said combination therapy.
 21. The methodaccording to claim 20, wherein said determining step comprises combiningsaid sample of cells with a binding agent that specifically recognizesp21^(waf1/cip1) protein.
 22. The method according to claim 21, whereinsaid binding agent is an antibody.
 23. The method according to claim 20,wherein said determining step comprises combining said sample of cellswith a binding agent that specifically recognizes p21^(waf1/cip1) mRNAor a cDNA derived therefrom.
 24. The method according to claim 20,further comprises the step of exposing said cells to a candidateanti-proliferative agent, and wherein said determining step comparesexpression of p21^(waf1/cip1) in the absence and presence of said agent.25. The method of claim 24, wherein said anti-proliferative agent is atopoisomerase inhibitor.
 26. The method of claim 25, wherein saidtopoisomerase inhibitor is an anthracycline.
 27. The method according toclaim 26, wherein said anthracycline is selected from the groupconsisting of doxorubicin, daunorubicin, epirubicin, idarubicin,anamycin, and MEN
 10755. 28. The method according to claim 24, whereinsaid anti-proliferative agent is CPT-11 (Irinotecan).
 29. The methodaccording to claim 24, wherein said anti-proliferative agent isvinorelbine.